Detection of infectious agents from environmental air dust

ABSTRACT

Embodiments of the present disclosure are directed to systems and methods for collection and analysis of environmental air dust (EAD) within an individually ventilated cage rack (IVR) environment for detecting pathogens. The method includes collection of an EAD sample by a collection media, isolation of a plurality of nucleic acids (e.g., RNA and/or DNA) representative of one or more infectious agents from the EAD sample, optional reverse transcription of RNA to cDNA if the isolated nucleic acids contain RNA, amplification of the cDNA and/or DNA (e.g., by polymerase chain reaction (PCR)), and assay interpretation. Optionally, the EAD sample may be analyzed with one or more other sample types (e.g., fecal pellets, oral swabs, body swabs, tissue, etc.) to improve detection of low-copy organisms.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/169,438, filed on Jun. 1, 2015, entitled “In-Line Airflow FiltrationFor Detection Of Infectious Agents” and U.S. Provisional Application No.62/280,057, filed on Jan. 18, 2016, and entitled “Method And System ForMonitoring Air Flow Impurity.” The entire teachings of each of the aboveapplications are incorporated herein by reference.

BACKGROUND

A requirement in biomedical research is that animals under study,typically mice, are monitored to confirm that they are free frominfection with specific pathogens. In one example, pathogens caninterfere with research by modulating experimental responses andcontaminating biologics, even if they rarely produce disease. In anotherexample, monitoring is needed to prepare current health reports tofacilitate shipment to and from collaborating institutions. In a furtherexample, institutional and/or governing bodies often require thatperiodic monitoring be performed.

In order to eliminate and then exclude pathogens, study animals aretypically re-derived or cured of infection and housed behind room- orcage-level barriers, respectively. These micro-isolation cage systemshave been widely adopted for maintaining and quarantining animals suchas mice and rats because the cage-level barrier they provide has provento be very effective at excluding and impeding the spread ofadventitious agents. No barrier can be guaranteed 100% effective,however. Thus, routine health monitoring (HM) is still necessary inmicro-isolation environments to verify the specific pathogen free (SPF)status of breeding and research colonies and imported animals inquarantine.

As study animals (e.g., rodents from research colonies) are rarely madeavailable to be bled or euthanized for conventional HM, they aretypically monitored indirectly through the use of soiled beddingsentinels (SBS). Using a soiled bedding approach, sentinel animals arehoused in a plurality of cages separate from the study animals andregular changes of soiled bedding pooled from the cages of study animalsare supplied to the sentinel cages. Over time, it is expected thatinfectious agents carried by the study animals are transferred to thepooled bedding and then to the sentinel animals. Thus, periodicscreening of the soiled bedding sentinels (e.g., pathology,parasitology, bacteriology, serology, etc.) provides indirect monitoringof the health of the study animals.

Reliance on soiled bedding sentinels for routine health monitoring hasbeen called into question in recent years, however. It is recognizedthat the switch to micro-isolator cages has been beneficial for limitingthe transmission of pathogens but presents a poor environment fordiagnostic monitoring. Notably, many agents are transmittedinefficiently, or not at all, to sentinel animals from study animals viasoiled bedding. Thus, as the prevalence of infection is lowered withinthe micro-isolator caging, the greater the risk that the pathogen dosein pooled bedding will be insufficient to infect sentinel animals.

For at least these reasons, there exists a continued need for newsystems and methods for monitoring study animals in micro-isolator cageenvironments.

SUMMARY

In an embodiment, a method for detecting pathogens is provided. Themethod includes receiving a test sample including environmental air dustcaptured from airflow within an exhaust plenum of an individuallyventilated cage rack (IVR) by a collection media, where a surface of thecollection media is oriented at an acute angle with respect to adirection of airflow within the exhaust plenum during capture of thedust sample. The method further includes isolating a plurality ofnucleic acids from the test sample, where the plurality of nucleic acidsis representative of one or more pathogens, amplifying at least one ofthe plurality of nucleic acids, and analyzing the amplified nucleicacids to identify the presence or absence of a pathogen.

In further embodiments, the method includes one or more of thefollowing, in any combination.

In an embodiment of the method, amplifying at least one of the pluralityof nucleic acids includes at least one of loop mediated isothermicamplification and polymerase chain reaction (PCR).

In an embodiment of the method, amplifying at least one of the pluralityof nucleic acids includes PCR selected from the group consisting ofendpoint PCR and real-time PCR.

In an embodiment of the method, isolating the plurality of nucleic acidsincludes extracting an RNA sample from the test sample and reversetranscribing the extracted RNA sample into a cDNA sample, amplifying atleast one of the plurality of nucleic acids includes amplifying the cDNAsample by real-time PCR, and analyzing the amplified nucleic acidsincludes measuring a Ct value of the amplified cDNA sample.

In an embodiment of the method, isolating the plurality of nucleic acidsincludes extracting a DNA sample from the test sample, amplifying atleast one of the plurality of nucleic acids includes amplifying the DNAsample by real-time PCR, and analyzing the amplified nucleic acidsincludes measuring a Ct value of the amplified DNA sample.

In an embodiment of the method, amplifying at least one of the pluralityof nucleic acids includes PCR and analyzing the amplified nucleic acidsincludes time of flight analysis of PCR products.

In an embodiment of the method, the test sample includes environmentalair dust captured from the airflow over a time period of at least 2weeks.

In an embodiment of the method, the IVR further includes at least onecage housing a test animal.

In an embodiment of the method, the test sample further includes atleast one of fecal pellets obtained from the test animal, biologicalmaterial obtained from an oral swap of the test animal, biologicalmaterial obtain from a body swab of the test animal, and tissue from thetest animal.

In an embodiment of the method, the one or more pathogens is selectedfrom the group consisting of: Staphylococcus spp., Pasteurella spp.,Proteus spp., Klebsiella spp., Giardia spp., Cryptosporidium spp.,Entamoeba spp., Spironucleus spp., Murine norovirus, Pseudomonas spp.,and beta-hemolytic Streptococcus spp.

In an embodiment of the method, isolating the plurality of nucleic acidsincludes at least one of magnetic isolation, column-based nucleic acidisolation, organic extraction methods, and alkaline lysis.

In an embodiment of the method, the IVR does not include a cage housinga sentinel animal.

In an embodiment of the method, the collection media includes a filterselected from the group consisting of mechanical filters, chemicalfilters, electrostatic filters, and wet scrubbers.

In an embodiment of the method, the collection media possesses anefficiency selected within the range between 5% to 40%.

In an embodiment of the method, the collection media is a graded filter.

In an embodiment of the method, the angle of the collection media duringcapture of the dust sample is selected from the range of 15° to 25° withrespect to the direction of airflow within the exhaust plenum.

In another embodiment, a method for detecting pathogens is provided. Themethod includes receiving a test sample including environmental air dustcaptured from airflow passing through an enclosure by a collectionmedia, the enclosure including a chamber containing an animal cage influid communication with the airflow and the environmental air dustreleased by agitation of soiled bedding of a test animal positionedwithin the animal cage. The method further includes isolating aplurality of nucleic acids from the test sample, where the plurality ofnucleic acids is representative of one or more pathogens, amplifying atleast one of the plurality of nucleic acids, and analyzing the amplifiednucleic acids to identify the presence or absence of a pathogen.

Further embodiments of the method may include one or more of thefollowing, in any combination.

In an embodiment of the method, amplifying at least one of the pluralityof nucleic acids includes at least one of loop mediated isothermicamplification and polymerase chain reaction (PCR).

In an embodiment of the method, amplifying at least one of the pluralityof nucleic acids includes PCR selected from the group consisting ofendpoint PCR and real-time PCR.

In an embodiment of the method, isolating the plurality of nucleic acidsincludes extracting an RNA sample from the test sample and reversetranscribing the extracted RNA sample into a cDNA sample, amplifying atleast one of the plurality of nucleic acids includes amplifying the cDNAsample by real-time PCR, and analyzing the amplified nucleic acidsincludes measuring a Ct value of the amplified cDNA sample.

In an embodiment of the method, isolating the plurality of nucleic acidsincludes extracting a DNA sample from the test sample, amplifying atleast one of the plurality of nucleic acids includes amplifying the DNAsample by real-time PCR, and analyzing the amplified nucleic acidsincludes measuring a Ct value of the amplified DNA sample.

In an embodiment of the method, amplifying at least one of the pluralityof nucleic acids includes PCR and analyzing the amplified nucleic acidsincludes time of flight analysis of PCR products.

In an embodiment of the method, the test sample includes environmentalair dust captured from the airflow over a time period of at least 2weeks.

In an embodiment of the method, the environmental air dust is releasedby agitation of the soiled bedding by a test animal.

In an embodiment of the method, the environmental air dust is releasedby an actuation device in mechanical communication with the enclosure.

In an embodiment of the method, the enclosure does not contain an animalduring capture of the environmental air dust.

In an embodiment of the method, the test sample further includes atleast one of fecal pellets obtained from the test animal, biologicalmaterial obtained from an oral swap of the test animal, biologicalmaterial obtain from a body swab of the test animal, and tissue from thetest animal.

In an embodiment of the method, the one or more pathogens is selectedfrom the group consisting of: Staphylococcus spp., Pasteurella spp.,Proteus spp., Klebsiella spp., Giardia spp., Cryptosporidium spp.,Entamoeba spp., Spironucleus spp., Murine norovirus, Pseudomonas spp.,and beta-hemolytic Streptococcus spp.

In an embodiment of the method, isolating the plurality of nucleic acidsincludes at least one of magnetic isolation, column-based nucleic acidisolation, organic extraction methods, and alkaline lysis.

In an embodiment of the method, the collection media includes a filterselected from the group consisting of mechanical filters, chemicalfilters, electrostatic filters, and wet scrubbers.

In an embodiment of the method, the collection media possesses anefficiency selected within the range between 5% to 40%.

In an embodiment of the method, the collection media is a graded filter.

In a further embodiment, a system for collecting environmental air dustis provided. The system includes a reversibly sealable enclosure. Theenclosure includes a chamber adapted to receive a cage for housing ananimal, the cage being in fluid communication with the chamber, an airintake coupleable with an air supply, and an air return coupleable witha vacuum source, where a portion of a flow of supplied air directedthrough the chamber, from the air supply to the air exhaust, passesthrough at least a portion of the cage when received in the chamber andis sufficient to transport a portion of environmental air dust containedwithin the received cage to the air exhaust. The system further includesan actuation device in mechanical communication with the enclosure, theactuation device operable to agitate the contents of the cage whenreceived within the chamber, and a collection media suitable forcapturing at least a portion of the environmental air dust transportedby a flow of air directed through the chamber.

Embodiments of the system may include one or more of the following, inany combination.

In an embodiment of the system, the collection media is positioned withrespect to the enclosure such that the collection media impinges atleast a portion of the flow of air after passage through the cage.

In an embodiment of the system, the collection media is positionedwithin the enclosure and outside of the cage.

In an embodiment of the system, the collection media is positioned withrespect to the enclosure such that the collection device impinges atleast a portion of the flow of air after passage through the air return.

In an embodiment of the system, at least a portion of the collectionmedia is positioned on a wall of the cage.

In an embodiment of the system, at least a portion of the collectionmedia is suspended within the cage.

In an embodiment of the system, the collection media includes a filterselected from the group consisting of mechanical filters, chemicalfilters, electrostatic filters, and wet scrubbers.

In an embodiment of the system, the actuation device is operable toreversibly move the enclosure at least one of translationally androtationally with respect to an initial position.

In an embodiment of the system, the actuation device is an ultrasonicdevice.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, features and advantages will beapparent from the following more particular description of theembodiments, as illustrated in the accompanying drawings in which likereference characters refer to the same parts throughout the differentviews. The drawings are not necessarily to scale, emphasis instead beingplaced upon illustrating the principles of the embodiments.

FIG. 1 is a schematic flow diagram illustrating an embodiment of amethod for collecting and analyzing Environmental Air Dust (EAD) from anindividually ventilated cage rack (IVR) for identification of pathogenscontained therein;

FIG. 2 is a schematic illustration of placement of a plurality of EADcollection media within an IVR environment;

FIG. 3A is a schematic illustration of placement of a plurality of EADcollection media within an IVR environment;

FIG. 3B is a schematic illustration of an embodiment of a cage includingan EAD collection media positioned on-cage for use in collecting EADfrom the cage within an IVR environment;

FIGS. 4A, 4B, and 4C illustrate embodiments of an EAD collection mediaholder for use within the IVR environment of FIG. 3A;

FIG. 4D is a photograph illustrating placement of an EAD collectionmedia in a vertical exhaust plenum of the IVR;

FIG. 4E is a photograph illustrating placement of an EAD collectionmedia adjacent a pre-HEPA filter in fluid communication with the IVR;

FIGS. 5A, 5B, and 5C illustrate a second embodiment of an EAD collectionmedia holder for use within the IVR environment of FIG. 3A;

FIGS. 6A and 6B are schematic illustrations of embodiments of EADcollection media;

FIGS. 7A and 7B are photographs illustrating locations from which EADsamples are collected by swab for comparative analysis; (A) Horizontalexhaust plenum; (B) Exhaust plenum hose;

FIGS. 8A, 8B, 8C, and 8D are plots comparing estimated copy of pathogensdetected by EAD collected from the EAD collection media and swabs; and

FIG. 9 is a schematic illustration of an embodiment of an EAD collectionsystem configured for operation independent of an individuallyventilated cage rack.

DETAILED DESCRIPTION

Embodiments of the present disclosure are directed to systems andmethods for collection and analysis of environmental air dust (EAD). Incertain embodiments, the systems and methods may be employed incombination with micro-isolator caging, such as static micro-isolatorcages and individually ventilated cages (IVC) mounted in a rack. Inalternative embodiments, the systems and methods may be employed incombination with non-IVC racks (e.g., open top or open cage racks wherethere is no air flow to accommodate collection of EAD).

Pathogen molecules, whether whole or fragments of a whole, are capableof attachment to air dust. This air dust, in turn, may be suspended in aflow of air and moved from one location to another. Thus, EAD collectedfrom airflow passing through a pathogen rich environment may be analyzedto detect the presence of such pathogens within the cage environment.

In an embodiment, a method of detecting pathogens carried byenvironmental air dust includes collection of a test sample includingEAD, isolation of a plurality of nucleic acids (e.g., RNA and/or DNA)representative of one or more infectious agents from the test sample,optional reverse transcription of RNA to cDNA if the isolated nucleicacids contain RNA, amplification of the cDNA and/or DNA (e.g., bypolymerase chain reaction (PCR)), and assay interpretation. Optionally,as discussed below, the test sample may include biological materialother than that captured by the collected EAD sample (e.g., fecalpellets, body swabs, oral swabs, tissue, etc.) to improve detection oflow-copy organisms. In such circumstances, pre-processing of the non-EADsample may be performed prior to nucleic acid co-isolation with the EADsample.

The EAD sample is collected from a target environment such as anindividually ventilated cage rack (IVR). In an IVR, a flow of sterileair is supplied to a first end of the rack and directed through cages ofthe rack (e.g., horizontally) to an opposing second end of the rack.EAD, and pathogens attached thereto, are carried by the airflow fromtheir respective cages out of the rack. A plurality of EAD collectionmedia, suitable for capturing at least a portion of the EAD, arepositioned in the airflow path. Thus, during use of the ventilated rack,the plurality of EAD collection media impinge at least a portion of theairflow, collecting EAD and attached pathogens from the cages.

In one embodiment, an EAD collection media is positioned within anexhaust channel that routes exhaust airflow away from the cages. Inanother embodiment, an EAD collection media is positioned on, adjacent,and/or within one or more cages.

In a further embodiment, a method of detecting pathogens carried byenvironmental air dust does not include collection of EAD within anindividually ventilated cage rack (IVR) system. Instead, the EAD iscollected from soiled bedding outside of an IVR (e.g., bedding soiled bya test animal). For example, in an embodiment, the method includescollecting EAD from a reversibly sealable enclosure housing an animalcage containing soiled bedding. The enclosure includes a chamber adaptedto receive the animal cage, an air intake coupleable with an air supply,and an air return coupleable with a vacuum source. In use, a flow of airis provided through the enclosure, from the air intake to the airreturn. The soiled bedding is agitated to free dust containing pathogensinto the air within the cage. In one embodiment, the bedding may beagitated by an animal placed within the cage (e.g., the test animal). Inanother embodiment, the enclosure is in communication with an actuationdevice which agitates the cage. The released EAD and attached pathogensare carried by the airflow out of the cage.

The plurality of EAD collection media are positioned so as to impinge atleast a portion of the flow of air, allowing collection of EAD andattached pathogens. In one embodiment, an EAD collection media ismounted outside of the enclosure, within an exhaust channel that routesexhaust airflow away from the enclosure. In another embodiment, an EADcollection device is mounted on, adjacent to, and/or within the cagehoused within the enclosure chamber.

EAD captured by the collection media may be subsequently analyzed toidentify the presence of selected pathogens. This EAD analysis may beperformed by techniques including, but not limited to, polymerase chainreaction (PCR) as noted above and discussed in greater detail below.

Notably, embodiments of the disclosed systems and methods may beemployed to sample airborne pathogens attached to air dust from theenvironment of study animals directly, rather than requiring the use ofsentinel animals. Beneficially, eliminating the use of sentinel animalsavoids sentinel husbandry and the need for weekly bedding transfers, aswell as saves rack space. Additionally, as shown in greater detailbelow, embodiments of the disclosed methods are capable of detectinginfectious agents that do not transfer efficiently to bedding sentinels,as well as those which do transfer efficiently to bedding sentinels.

Embodiments of the disclosure will now be discussed with respect to thefigures, beginning with FIGS. 1 and 2. FIG. 1 is a flow diagram of anembodiment of a method 100 for collecting and detecting pathogenscarried by environmental air dust (EAD). As discussed in detail below,the method 100 includes receiving a test sample including environmentalair dust in operation 102, isolating a plurality of nucleic acids (e.g.,RNA and/or DNA) representative of one or more infectious agents from theEAD sample in operation 104, optional reverse transcription of RNA tocDNA if the isolated nucleic acids contain RNA in operation 106,amplification of cDNA and/or DNA in operation 110 (e.g., by polymerasechain reaction (PCR)), and assay interpretation in operation 112. Incertain circumstances, the method 100 may optionally include samplepre-processing in operation 114.

FIG. 2 is a schematic illustration of an embodiment of a micro-isolatorcage environment 200 from which the received EAD is collected. For thepurpose of discussion, embodiments of the disclosure will be describedin the context of an individually ventilated cage rack (IVR) 202 as themicro-isolator cage environment 200. However, it may be understood thatthe disclosed embodiments may be employed with any micro-isolator cageswithout limit.

The IVR 202 includes a plurality of cages 208 for housing animals (e.g.,rodents) in a selected arrangement (e.g., an array having generallyaligned rows and columns). The cage environment 200 further includes aventilation system 204 in fluid communication with the IVR 202 via anairflow network 206. The ventilation system 204 includes an air handlingunit 210 that provides sterile, conditioned supply air 212 to an airintake 214 of the IVR 202 and receives exhaust air 216 (e.g., airincluding dust and attached pathogen molecules) from an air return 220of the IVR 202. In the IVR 202, the supply air 212 is distributed todifferent locations (e.g., different rows and/or columns) and flowsthrough each cage 208. This airflow within the IVR 202, referred to asIVR airflow 222, is sufficient to transport at least a portion of EADfrom respective cage locations through the air return 220. From the airreturn 220, airflow including the EAD travels to the air handling unit210 and is referred to as exhaust air 216 herein.

The embodiment of FIG. 2 is illustrated with a closed ventilationsystem, where the exhaust air 216 is reconditioned (e.g., to a selectedtemperature, pressure, sterility, etc.) by the air handling unit 210 andsubsequently provided to the IVR 202 as the supply air 212. However, inalternative embodiments, not shown, the ventilation system may be anopen system, where the air supply is not provided from reconditioned airexhaust but is instead provided from a separate source.

A plurality of EAD collection media 224 (e.g. 224 a, 224 b) are furtherprovided for capturing a portion of EAD within the IVR 202, whileallowing the supply air 212, IVR airflow 222, and exhaust air 216 tomove through the IVR 202 substantially unimpeded. Captured EAD may beretained on the surface of the EAD collection media 224, the bulk of theEAD collection media 224, and/or within a dedicated containment chamber,based on the configuration of the EAD collection media 224. Example EADcollection media 224 may include, but are not limited to, mechanicalfilters, chemical filters, wet scrubbers, electrostatic filters, andother filtering devices suitable for removing dust from air, withoutlimit.

Each EAD collection media 224 may be independently selected to captureEAD having a size (e.g., diameter, cross-sectional area, or otherselected dimension) approximately greater than a selected value. Inother embodiments, the EAD collection media 224 may be graded (e.g., agraded filter), with spatial regions selected to capture EAD ofdifferent sizes. Such zones may vary continuously or discontinuouslywithin the spatial extent of the EAD collection media 224. In certainembodiments, EAD collection media 224 may be selected to capture dustparticles having a size ranging from about 0.1 nm to about 10 mm. Infurther embodiments, EAD collection media 224 may possess efficiencyselected within the range between about 5% to about 40% for the selectedsize range. In certain embodiments, the efficiency of EAD collectionmedia 224 may be approximately 30% for the selected size range. Furtherdiscussion of EAD collection media 224 may be found within U.S.Provisional application No. 62/280,057, filed on Jan. 18, 2016 andentitled “Method And System For Monitoring Air Flow Impurity,” theentirety of which is incorporated by reference.

The position of the EAD collection media 224 may be varied, dependingupon the configuration of the IVR 202. For example, as illustrated inFIG. 2, one or more EAD collection media 224 a may be positioned withina portion of the airflow network 206 to capture EAD from the exhaust air216. In alternative embodiments, one or more EAD collection media 224 bmay be positioned on or within one or more selected cages 208 to captureEAD from the IVR airflow 222. In further embodiments, at least one EADcollection media 224 may be positioned to capture EAD from both theexhaust air 216 and the IVR airflow 222.

Regardless of position, each the EAD collection media 224 is maintainedin place for a selected time duration to capture EAD thereon. In certainembodiments, the selected time duration may be a minimum of 2 weeks. Infurther embodiments, the selected time duration may be 3-4 months. Infurther embodiments, the selected time duration may be one year or more.

In order to maintain sufficient ventilation within the IVR 202, the EADcollection media 224 may further deployed in a manner which does notsubstantially interfere with airflow within the ventilation system 204(e.g., supply air 212, IVR airflow 222 and/or exhaust air 216). Forexample, as discussed in greater detail below with respect to FIGS. 3and 4, the EAD collection media 224 may be understood to notsubstantially interfere with airflow when at least one of the followingconditions are satisfied:

(i) a blower motor of the ventilation system 204 does not exhibit achange in RPM (speed) due to increased pressure caused by EAD collectionmedia 322 (e.g. 322 a, 322 b, 322 c) or the presence of an EADcollection media holder 400 (FIGS. 4A-4C) maintaining EAD collectionmedia 322 (e.g., 322 a) within a vertical exhaust plenum 316 (FIG. 4D);

(ii) a pressure within one or more cages 308 (FIG. 3A) varies by lessthan or equal to ±5% due to reduced airflow caused by a restriction inthe vertical exhaust plenum 316 due to the presence of the EADcollection media 322 a and/or the EAD collection media holder 400 ascompared to the pressure within the one or more cages 308 absent the EADcollection media 322 a and/or the EAD collection media holder 400 withinthe vertical exhaust plenum 316;

(iii) a pressure within the vertical exhaust plenum 316 (e.g., at anexhaust collar or terminal end 450 of the vertical exhaust plenum 316)varies by less than or equal to ±10% due to the presence of the EADcollection media 322 a and/or the EAD collection media holder 400 withinthe vertical exhaust plenum 316 as compared to the pressure within thevertical exhaust plenum 316 absent the EAD collection media 322 a and/orthe EAD collection media holder 400.

In further embodiments, the configuration of the EAD collection media224 and/or the positioning of the EAD collection media 224 within theIVR 202 facilitates maintenance of sufficient ventilation within the IVR202. In one embodiment, respective dimensions of the EAD collectionmedia 224 may be less than (e.g., 30% less, 40% less, 50% less, 60%less, 70% less, etc.) than corresponding dimensions of an airflowpassageway in which the EAD collection media 224 is positioned oradjacent to (e.g., vertical exhaust plenum 316, pre-HEPA filter 450,etc.). In another embodiment, described in greater detail below, the EADcollection media 224 may be placed such that the plane of the EADcollection media 224 is oriented at an acute angle with respect to adirection of airflow (e.g., vertical airflow within the vertical exhaustplenum 316) during capture of the dust sample. The acute angle may beselected within the range from 15° to 25° (e.g., 20°) with respect tothe direction of airflow within the vertical exhaust plenum 316.

Following collection of EAD by capture using the EAD collection media224, at least a portion of the collected EAD is analyzed to detect thepresence or absence of a pathogen in operations 104-112. To minimizehandling and contamination of the EAD, and facilitate transport, the EADcollection media may be removed from the IVR environment 200 anddirectly placed in a sterile container (e.g., a reversibly sealabletube).

In certain embodiments of the method, receiving a test sample includingcaptured EAD may include receiving the captured EAD itself. In otherembodiments, receiving a test sample including captured EAD may includereceiving the captured EAD while still retained on the EAD collectionmedia. In further embodiments, receiving a test sample includingcaptured EAD may include collecting the EAD from airflow, as discussedherein.

With continued reference to FIG. 1, certain embodiments of the method100 may include a pre-processing operation 114 for preparation of anon-EAD sample to be analyzed with collected EAD samples. Examples ofsuch non-EAD samples may include, but are not limited to, one or more offecal pellets, biological material collected from oral swabs, biologicalmaterial collected from body swaps, and tissue.

For example, assume that the non-EAD sample is fecal pellets. Thepre-processing operation 114 processes the fecal pellets into a slurryfor later use in isolating nucleic acids for testing. Fecal pellets maybe collected from rack cages. In further embodiments, fecal pellets maybe obtained from different rodent lines and adult rodents in targetoptimal age ranges (e.g., 6-10 weeks). Once obtained, one or more of thefecal pellets are transferred to a micro-centrifuge tube with one ormore stainless steel ball bearings. A selected amount of phosphatebuffered saline (PBS) is added to the tube containing the fecalpellet(s) to form a fecal pellet sample. The fecal pellet sample isdisrupted in a mixer mill for a selected time at a selected frequencyand subsequently centrifuged. Subsequently, the fecal sample may bepooled with collected EAD for nucleic acid co-extraction.

Because fecal pellets contain bile acids, hemoglobin breakdown products,complex polysaccharides, and other compounds that may inhibitamplification, the pre-processing operation 114 is performed toconcentrate agents of interest (e.g., viruses, bacteria, and othermicro/macrobiological material associated pathogens) for nucleic acidisolation, yet also excluding as much as the fecal content as possible.

Because the pre-processing operation 114 concentrates agents ofinterest, performing pre-processing operation 114 as part of the method100 may be beneficial to improve detection of low copy organisms.Examples of low copy organisms may include, but are not limited to,Staphylococcus spp. (bacteria), Pasteurella spp., Proteus spp.,Klebsiella spp. (bacteria), Giardia spp. (protozoa), Cryptosporidiumspp. (protozoa), Entamoeba spp. (protozoan), Spironucleus spp.(protozoan), Murine norovirus (virus), Pseudomonas spp., andbeta-hemolytic Streptococcus spp.

In operation 104, the test sample including collected EAD, optionallypooled with non-EAD material pre-processed in operation 114, is furtherprocessed to isolate nucleic acids therefrom (e.g., RNA, DNA, andcombinations thereof). In certain embodiments, the nucleic acidisolation is performed by magnetic isolation. Magnetic isolation,although limited in the total yield, does provide a highly purifiednucleic acid. In the inventor's experience, magnetic isolation includesbetter recovery of RNA versus other column methods as well as anobserved reduction of PCR inhibitors. However, it may be understood thatalternative processes for nucleic acid isolation may be employed, aloneor in combination with magnetic isolation, without limit. Examples ofsuch alternative isolation processes include, one or more ofcolumn-based nucleic acid isolation, organic extraction methods, andalkaline lysis.

In further embodiments, a nucleic acid recover control (NARC) may beoptionally added to the isolated nucleic acids in operation 104. TheNARC is a unique algae RNA (or DNA when only DNA tests are performed).By adding the NARC to the lysis buffer, it allows monitoring of anysample type and provides verification that any nucleic acid present atthe lysis step was successfully recovered. Some samples may containlittle or no nucleic acid and others may have great variation, someasuring optical density to determine nucleic acid content is notuseful. Accordingly, successful extraction (recovery of nucleic acid) aswell as a successful reverse transcription reaction are each verified byperforming a separate assay for the NARC template as part of thecontrols run during the amplification test analysis.

In operation 106, when RNA is present, alone or in combination with DNA,the RNA is reverse transcribed into cDNA, which is required for RNAviruses. Alternatively, if only DNA is present in the isolated nucleicacid, this reverse transcription operation may be omitted.

Following reverse transcription, amplification is performed in operation110. In an embodiment, amplification may be performed by loop mediatedisothermic amplification or polymerase chain reaction (PCR). Examples ofPCR include, but are not limited to, endpoint PCR (e.g., agaroseelectrophoresis, hybridization arrays, etc.) and real-time PCR (e.g.,molecular beacon PCR, TaqMan PCR, SYBR Green PCR, etc.).

In an embodiment, the amplification is performed by TaqMan PCR. Samplesthat are evaluated for small panels of agents are evaluated on a 96-wellor 384-well real-time TaqMan PCR platform. Samples that are evaluatedfor large panels of agents are first pre-amplified then amplified byreal-time TaqMan PCR on an open array. A DNA spike control to monitorfor PCR inhibition is also added at the pre-amplification step forpanels evaluated on the open array. For 96 and 384-well formats, thespike is added as a separate assay during the PCR testing for allsamples.

A PCR Assay is subsequently performed for detection of selectedinfectious agents. Approximately 100 copy templates are prepared bycloning target sequences into plasmid vectors and used as positivecontrol templates. About 1000 copy plasmids are also included for openarray testing. Negative template wells are used for all formats. NARCand spike control mock samples processed alongside of field samples areevaluated to demonstrate function of these control templates and assays.

In operation 112, an analysis of the assay is performed. Following PCRamplification, Ct values (threshold cycle) are measured for samples andthe samples are evaluated based upon the measured Ct values. The Ctvalues are a relative measure of the concentration of target in the PCRreaction. Further information regarding specimen analysis may be foundin “Efficacy of Direct Detection of Pathogens in Naturally Infected Miceby Using a High-Density PCR Array,” J. Am Assoc. Lab Anim. Sci. Vol. 52,No. 6, pgs. 763-772 (2013), the entirety of which is incorporated hereinby reference.

Examples

In the following examples, low prevalence infection was simulated on anIVR. Samples of EAD were collected by use of collection devices in-linewith airflow. Comparative samples were collected from soiled beddingsentinels and swabs. All collected samples were separately analyzedusing PCR according to embodiments of the method 100 of FIG. 1 discussedabove. Samples obtained from soiled bedding sentinels were furtheranalyzed by traditional screening (e.g., serology, bacterial culture,parasitology).

Methodology Study Design

Infectious Agent Source:

Pet shop mice were placed in 4 cages on each rack side. Two mice werehoused in each cage, one 3-4 weeks old and the other 6-10 weeks old.Assuming 80 total cages on the rack, these conditions simulated aprevalence of about 5% on rack (4/80 cages).

Sentinel Animals:

4 cages of CD-1 Elite bedding sentinels were provided on the IVR. 3sentinel mice were provided per cage. 1 mouse per cage per month wassubmitted for PCR and traditional testing (total of 4 per month).

Simulation of a Low Prevalence:

5% “true dilution” soiled bedding from the pet shop mice was placed withthe sentinel mice. The soiled bedding was diluted with soiled beddingfrom gnotobiotic mice to achieve the desired concentration.

All cages were placed in furthest locations from incipient EAD swabsites.

Exhaust Air Dust Sample Collection

With reference to FIGS. 3A-3B and 4A-4C, collection of EAD samples byin-line EAD collection media and swab will now be discussed. FIG. 3Apresents a schematic illustration of an embodiment of a cage environment300 including an IVR 302 housing cages 308 in fluid communication with aclosed ventilation system 304 from which EAD is collected in the instantexamples. A supply of sterile air 312 is delivered to a generallyvertical supply plenum 306 of the IVR 302 from the ventilation system304. The vertical supply plenum 306 directs the sterile supply air 312through the cages 308 via a plurality of generally elongated horizontalplenums 314 from the vertical supply plenum 306 to an opposing secondend of the rack including a generally vertical exhaust plenum 316. EADand pathogens attached thereto are carried by IVR air flow 320 throughrespective cages 308 out of the IVR 302 as exhaust air 320. Theventilation system 304 includes a pre-HEPA filter assembly 310 a and aHEPA filter assembly 310 b. The pre-HEPA filter assembly 310 a receivesthe exhaust air 320. Air flowing through the pre-HEPA filter assembly310 a is directed into the HEPA filter assembly 310 b, where it exits assupply air 312.

A plurality of EAD collection media 224 are further positioned tocapture EAD while not interfering with airflow through the IVR 302. Afirst EAD collection media 322 a is a physical filter suspended near atop of the vertical exhaust plenum 316, referred to herein as exhaustplenum filter 322 a.

With further reference to FIGS. 4A-4D, the exhaust plenum filter 322 ais supported within the vertical exhaust plenum 316 by an EAD collectionmedia holder 400. The holder 400 includes a ring collar 402 attached toa frame 410. As discussed in greater detail below, in use, the ringcollar 502 can be slip fit into a terminus 450 of the vertical exhaustplenum 316.

In an embodiment, the holder 400 and frame 402 can be formed of metal orplastic. In one embodiment, the frame 402 is formed of stainless steeland the ring collar 402 is welded to the frame 402. The frame 404 can beformed of a plurality of horizontal members 406 extending from verticalmember 410 to form a grid pattern. Vertical lip 412 can be positioned atouter edges 414 of the frame 404. The grid pattern retains the EADcollection media 224 and provides sufficient contact of exhaust air 320with the exhaust plenum filter 322 a.

The frame 404 can be rotated in the directions of arrows 432 forpositioning the frame 404 an angle, A, with respect to exhaust air 320within the vertical exhaust plenum 316. This positioning tailors thedegree of contact of exhaust air 320 with the exhaust plenum filter 322a (e.g., flow of exhaust air 320 along the length of the exhaust plenumfilter 322 a). This adjustability is beneficial for minimizing theimpact of the exhaust plenum filter 322 a on airflow within the IVR 202.A horizontal lip 416 at an end 420 of the frame 404 can further generateturbulence within the exhaust air 320 flowing over collection mediaholder 404 to facilitate EAD capture from the exhaust air 320.

A spring clip 422 can be attached to center mount 424 of ring mounting112 by inserting an end 426 of the spring clip 422 through a slot 430 inthe center mount 424. The spring clip 422 secures the EAD collectionmedia 322 a to the frame 404. Moving the spring clip 422 away from theplane of the frame 404 allows insertion or removal of the exhaust plenumfilter 322 a from the EAD collection media holder 400.

FIGS. 5A-5C show an alternate embodiment of a EAD collection mediaholder 500 for support of exhaust plenum filter 322 a. Ring collar 502can be attached to a plurality of frames, such as 504 a, 504 b. The ringcollar 502 and frames 502 a, 502 b are as discussed previously withrespect to ring collar 402 and frame 404 except as outlined below. Ringcollar 502 can be slip fit into the terminus 450 of the vertical exhaustplenum 316. Each of the EAD collection media holders 504 a, 504 b can beangled at an angle A with respect to exhaust air 320 within the verticalexhaust plenum 316. This positioning allows the degree of contact ofexhaust air 320 with the exhaust plenum filter 322 a (e.g., flow ofexhaust air 320 along the length of the exhaust plenum filter 322 a) tobe adjusted. This adjustability is beneficial for minimizing the impactof the exhaust plenum filter 322 a on airflow within the IVR 202.

Spring clip 522 a can attach the EAD collection media holder 504 tocenter mount 524 a of ring mounting 502 by inserting end 526 a of springclip 522 a through slot 530 a in center mount 524 a. Spring clip 522 bcan attach the EAD collection media holder 504 b to center mount 524 bof ring collar 502 by inserting end 526 b of the spring clip 522 bthrough the slot 530 b in center mount 524 b. The spring clips 522 a,522 b provide clamping of EAD collection media 224 a as shown in FIG.5B. Moving the spring clip 522 a, 522 b away from the plane of theirrespective frame 504 a, 504 b allows insertion or removal of the EADcollection media 224 a from the EAD collection media holder 500.

FIGS. 6A-6B are schematic illustrations of embodiments of EAD collectionmedia 224 suitable for use one or more of EAD collection media (e.g.,224 a, 224 b, 322 a, 322 b, 322 c). EAD collection media 224 includesmedia 602 coupled to a media frame 604. The media frame 604 extendsaround an outer edge 606 of the media 602. In one embodiment, the media602 is heat sealed to the media frame 604 for provide a more rigidstructure that facilitates handling, insertion, and removal of the media602 (e.g., from EAD media holder 400, 500). The media frame 604 mayoptionally be marked with indicia 610 (e.g., a dot or notch) for use inidentifying a direction of insertion of the EAD collection media 224into EAD collection media holder 400, 500. The media frame 604 mayfurther include one or more chamfers 612 at positioned at an angle αwith respect to its longitudinal edges.

In an embodiment, the media 602 can be constructed of an electrostaticand controlled gradient structure including thermally wovenpolypropylene/polyethylene fibers, spun bond, continuous filament andattracts both positively and negatively charged particles with lowmoisture regain. The media 602 can prevent fibers from swelling due tomoisture absorbed from the air stream. In one embodiment, the media 602is white. The media 602 can carry a varying MERV (Minimum EfficiencyRating Value) rating between about 6 and about 10 (e.g., 8). In furtherembodiments, the media 602 may possess a basis weight of 2.00 oz/yd²,thickness 64 mils, and Frazier Air Permeability 490 cfm/ft². In otherembodiments, the polypropylene fibers of media 602 can be substituted bypolyolefin fibers.

In an embodiment, the frame 602 can be manufactured of various syntheticfibers (e.g., polyester fibers). In one embodiment, the frame 602 alsofunctions as a collection media. For example, the frame 602 may includea white spun collection media with a basis weight of 8.0 oz/yd², nominalthickness of 23 mils. strip tensile MD of 125 ft. lb/0.6 in. In furtherembodiments, the frame 602 can be constructed of any of collection mediathat can be heat sealed to EAD media 604 and has a basis weight between6 and 10.

FIG. 6B shows an alternative embodiment of the EAD collection media 224including a perforation 650. Perforation 650 is positioned between afirst side 652 and a second side 654 of EAD collection media 224. Theperforation 650 can be separated to separate the first and second sides652, 654 of EAD collection media 224 from one another.

In a non-limiting embodiment, the dimensions of the EAD collection media224 are as follows. A total length, L₁, of the EAD collection 224 is3.75 in. A length, L₂, from a first end of the media frame 604 tochamfer 612 is 3.25 in. A frame border, B, is 0.375 in. An EADCollection media width, W, is 2.312 in. An EAD collection mediathickness, T, is less than or equal to 0.032 in. It may be understoodthat the above dimensions are provided for the purpose of example andthat the EAD collection media 224 may possess other dimensions, asnecessary.

During EAD collection, the ring collar 402 or 502 is mounted adjacent atop end of the vertical exhaust plenum 316 (FIG. 4D). So positioned, theexhaust plenum filter 322 a is suspended within the vertical exhaustplenum 316 with angle A set as an acute angle between plane of theexhaust plenum filter 322 a and a flow direction of the exhaust air 320.In an embodiment, the acute angle is selected within the range of 15° to25° (e.g., 20°) with respect to the direction of airflow within thevertical exhaust plenum 316. In further embodiments, the in-planedimensions of the exhaust plenum filter 322 a may be also smaller thanthe cross-sectional dimensions of the vertical exhaust plenum 316. Inthis manner, EAD may be captured from the exhaust air 320 withoutsubstantially interfering with the flow of exhaust air 320.

As further illustrated in FIGS. 3A and 4C, EAD was also collected usinga second EAD collection media 322 b (a physical filter) attached to aleading face (upstream facing) of a pre-HEPA filter 450 of the pre-HEPAfilter assembly 310 a. Each of the EAD collection media 322 a, 322 bwere deployed for 3 months. As each of the EAD collection media 322 a,322 b capture EAD from the exhaust air 320 (i.e., air that has passedthrough the extent of the IVR 302), any EAD collected is expected to berepresentative of the IVR 302 as a whole.

As additionally illustrated in FIG. 3B, EAD was further collected fromairflow 350 within and/or adjacent cages 308 containing soiled bedding(not shown). Each cage 308 included a bottom 352, a top 354, sidewalls356, a porous lid 360, and a third EAD collection media 322 c. The thirdEAD collection media 322 a (a physical filter) was placed on the cagetop 356, which is in fluid communication through the cage lid 356 withairflow 350 a circulating through an interior volume of the cage 308 aswell as airflow 350 b passing over the cage lid 350. However, it may beunderstood that, in alternative embodiments, EAD collection media may bepositioned on any cage sidewall, within any cage, or adjacent to anycage, as appropriate. A new EAD collection media 322 c (2″ or 2.5″×3″)was placed on each cage top 360 at the beginning of the study and every3 months thereafter. The cage top 360 was further transferred each timethe cage 308 was cleaned.

Additional dust samples were obtained from swabs of the IVR 302 forcomparison with dust samples collected by the EAD collection media 322a, 322 b, 322 c. Dust swabs were collected at each horizontal plenum 314and pooled (FIG. 7A), with evaluation every 3 months. Two dust swabswere also collected from an exhaust hose 700 connecting the verticalexhaust plenum 316 to the pre-HEPA filter assembly 310 a (FIG. 7B), withevaluation every 3 months.

Sample Analysis

Nucleic Acid Isolation:

Nucleic acid isolation is performed by magnetic isolation. For EADcaptured by EAD collection media, the EAD collection media is positionedin a tube (e.g., the same tube in which the EAD collection media istransported), curved with the EAD facing inward. For dust collected byswabs, the swab(s) are transferred to a clean tube large enough toaccommodate the number of swabs to be analyzed. A lysis solution ispipetted directly onto the EAD collection media or swab(s).

Respective tubes are subsequently subjected to vortex to thoroughly washthe EAD collection media or swab(s) with the lysis solution. Respectivetubes are further centrifuged to spin the lysis solution down from thetop of the first tube. A nucleic acid recovery control (NARC) is furtheradded to respective tubes. The NARC allows for monitoring of any sampletype and provides verification of recovery of nucleic acids present atin the lysis. Successful extraction (recovery of nucleic acid) as wellas a successful reverse transcription reaction (RNA is transcribed tocDNA to detect RNA viruses by PCR) by performing a separate assay forthe NARC template as part of the controls run during the PCR testanalysis.

A selected amount of isopropanol is added to all sample wells, mixingwhile adding, followed by incubation at room temperature for at least 1minute. A uniform suspension of mixing beads is added to each wellcontaining a sample lysis and isopropanol.

Subsequently, the lysis samples and appropriate reagents are placed in aKingfisher™ Magnetic Particle Processor 96 (Thermo Scientific™, Waltham,Mass., USA). The Kingfisher™ uses permanent magnetic rods and disposabletip combs to move particles through the purification process.

Reverse Transcription:

Reverse transcription of RNA into cDNA is further performed if theisolated nucleic acids contain RNA, as cDNA is required for RNA viruses(PCR requires DNA as a template). The reverse transcription is performedusing a commercial kit. The kit reagents are prepared in reaction wellsaccording to the manufacturer's instructions. For each sample to betested that contains RNA, an appropriate volume of the sample is addedto the corresponding reaction well. The wells are covered andcentrifuged to ensure mixture of all components.

PCR Amplification:

Samples evaluated for small panels of infectious agents are on a 96-wellor 384-well real-time TaqMan PCR platform. Samples that are evaluatedfor large panels of infectious agents are first pre-amplified prior toamplification by real-time TaqMan PCR on an Open Array.

The pre-amplification employs a concentration of primers (the sameprimers used for TaqMan assays discussed below). It is observed that, atfull primer concentration, pre-amplification will permit an increase incopy number of about 3 Log 10. In general, primer concentration islimited to prevent any one template from dominating the reaction andpreventing detection of templates present in smaller copy number (primerfor large template agents is used up in the first couple cycles). Theprimer concentration is further limited in the pre-amplification forinfectious agents that are known to always be present in large copynumbers.

Spike Control—

A DNA spike control to monitor for PCR inhibition is added at thepre-amplification for panels evaluated on the Open Array. For 96 and384-well formats, the spike is added as a separate assay during the PCRtesting for all samples.

PCR Assays—

PCR assays have been designed to incorporate sequences from publicdomains and unique sequences not available in the public domain.

100 copy templates were prepared by cloning target sequences intoplasmid vectors and used as positive control templates. 1000 copyplasmids are also included for Open Array testing. Negative templatewells are used for all formats. NARC and spike control mock samplesprocessed alongside of field samples are evaluated to demonstratefunction of these control templates and assays.

Assay Interpretation:

Ct values obtained for samples are evaluated based on Ct value, but alsogeneral experience and observation to rule out cross-contamination.Valid CT values for determination of positive samples is based onexperience and general knowledge of individual agents.

Test Results Viruses

Table 1 compares testing results for samples obtained from fourdifferent sources: sentinel animals, EAD cage filters, EAD swabs, andEAD in-line filters. For sentinel animals, analysis is performed byserology, bacterial culture, and parasitology (“Traditional”) and PCR.For EAD swabs, samples acquired from hose and plenum swabs, as discussedabove, are analyzed by PCR. For EAD cage filters, dust samples areacquired without a sentinel animal being present within the cage (“noSentinel”) and with a sentinel animal present within the cage (“withSentinel”) and are analyzed by PCR. For EAD in-line filters, dustsamples acquired from EAD collection media positioned within the IVRvertical exhaust plenum and adjacent the pre-HEPA filter are analyzed byPCR.

TABLE 1 VIRUS TESTING RESULTS EAD Cage Filter EAD Swab EAD In-LineFilter Sentinel no with Hose Plenum Exhaust Pre-HEPA Agent TraditionalPCR Sentinel Sentinel Swab Swab Filter Filter Adenovirus 0 0 0 1 0 0 1 1Coronavirus 1 0 0 4 1 1 1 1 Mouse 4 1 0 4 1 1 1 1 ParvovirusesTheilovirus 0 0 1 2 1 1 1 1 Total % 31.3% 6.3% 6.3% 68.8% 75.0% 75.0%100% 100% PositiveFrom the results of Table 1, it may be observed that analysis of samplescollected from sentinel animals yield the poorest indicator of viruses,detecting only 31% of viruses by traditional screening and about 6.3% byPCR. Analysis of samples collected from the cage-level EAD collectionmedia exhibit mixed success, identifying only 6.3% of viruses when thesentinel was not present within the cage but approximately 68.8% ofviruses when the sentinel was present. Analysis of samples collectedfrom EAD swabs at the hose and plenum exhibit good accuracy, eachdetecting 75% of viruses. Analysis of samples collected in-line withexhaust air within the vertical exhaust plenum and adjacent the pre-HEPAfilter exhibit the best accuracy, each detecting 100% of the virusespresent.

Estimated copy of the above viruses, determined by real-time TaqMan PCRof dust samples collected from swabs and EAD collection media, isillustrated in FIG. 8A. As noted above, the Ct value is a relativemeasure of the concentration of a target. As elevated concentrationincreases likelihood of detection, higher values of Ct are preferred. Itmay be observed from FIG. 8A that for each virus, the EAD samplescollected by the EAD collection media demonstrate Ct values comparableto or greater than swabbing.

Bacteria

Sample analysis for selected bacteria is illustrated in Table 2 below.

TABLE 2 BACTERIA TESTING RESULTS EAD Cage Filter EAD Swab EAD In-LineFilter Sentinel no with Hose Plenum Exhaust Pre-HEPA Agent TraditionalPCR Sentinel Sentinel Swab Swab Filter Filter Beta Strep 0 0 0 0 0 0 1 1Group B H. ganmani N/A 0 0 2 0 0 1 H. hepaticus N/A 0 0 0 0 0 0 H.typhlonius N/A 0 0 3 1 1 1 Helicobacter N/A 0 2 4 1 1 1 M. pulmonis 0 30 2 1 1 1 1 P. mirabilis 0 0 2 3 1 0 0 0 P. multocida 0 0 0 1 0 0 1 1 P.pneumotropica 0 3 1 4 1 1 1 S. aureus 0 0 0 0 0 0 1 1 Total % 0% 15.0%12.5% 47.5% 50.0% 40% 80% 40% Positive

From Table 2, it may be observed that analysis of samples collected fromsentinel animals yield the poorest indicator of bacteria, detecting noneof bacteria by traditional screening and 15% by PCR. Analysis of samplescollected by the cage-level EAD collection media exhibit mixed success,identifying only 12.5% of bacteria when the sentinel is not presentwithin the cage but approximately 47.5% of bacteria when the sentinel ispresent. Analysis of samples collected from EAD swabs at the hose andplenum detect 50% and 40% of bacteria present. Analysis of samplescollected by EAD collection media in-line with exhaust air within thevertical exhaust plenum exhibit the best accuracy, detecting 80% of thebacteria present. Analysis of samples collected in-line with the exhaustairflow and adjacent the pre-HEPA filter detect 40% of the bacteriapresent.

Estimated copy of the above bacteria is determined by real-time TaqManPCR of dust samples collected from swabs and EAD collection media isillustrated in FIG. 8B. As noted above, the Ct value is a relativemeasure of the concentration of a target. As elevated concentrationincreases likelihood of detection, higher values of Ct are preferred. Itmay be observed from FIG. 8B that for each bacteria, the EAD samplescollected by the EAD collection media demonstrate Ct values roughlycomparable to or greater than swabbing.

Protozoa

Analysis of each sample for selected protozoa is illustrated in Table 3below.

TABLE 3 PROTOZOA TESTING RESULTS EAD Cage Filter EAD Swab EAD In-LineFilter Sentinel no with Hose Plenum Exhaust Pre-HEPA Agent TraditionalPCR Sentinel Sentinel Swab Swab Filter Filter Cryptosporidium 0 0 0 2 11 1 1 Entamoeba 2 2 0 4 1 1 1 1 Giardia 0 0 0 1 0 0 1 1 Spironucleusmuris 0 0 0 2 1 0 1 1 Tritichomonas 0 0 4 4 1 1 1 1 Total % Positive31.3% 10% 20% 65% 80% 60% 100% 100%From Table 3, it may be observed that analysis of samples collected fromsentinel animals yield relatively poor indication of protozoa, detectingonly 31.3% of protozoa by traditional screening and about 10% by PCR.Analysis of samples collected from the cage-level EAD collection mediaexhibit mixed success, identifying only 20% of protozoa when thesentinel was not present within the cage but approximately 65% ofprotozoa when the sentinel was present. Analysis of samples collectedfrom EAD swabs at the hose and plenum exhibit good accuracy, detecting80% of protozoa by hose swab and 60% of protozoa by plenum swab.Analysis of samples collected by EAD collection media in-line withexhaust airflow exhibit the best accuracy, each detecting 100% of theprotozoa present.

Estimated copy of the above protozoa determined by real-time TaqMan PCRof collected EAD samples is illustrated in FIG. 8C. As noted above, theCt value is a relative measure of the concentration of a target. Aselevated concentration increases likelihood of detection, higher valuesof Ct are preferred. It may be observed from FIG. 8C that for eachprotozoa, the EAD samples collected by the EAD collection mediademonstrated Ct values comparable to or greater than swabbing.

Metazoan Parasites

Analysis of each sample for selected parasites is illustrated in Table 4below.

TABLE 4 PARASITE TEST RESULTS Sentinel Cage Filter EAD Swab EAD In-LineSentinel no with Hose Plenum Exhaust Pre-HEPA Agent Traditional PCRSentinel Sentinel Swab Swab Filter Filter Demodex 0 0 0 1 0 1 1 1Myobia/Radforia 0 1 0 2 1 1 1 0 Myocoptes 0 0 0 0 1 1 1 0 Pinworms 0 0 04 0 1 1 1 Total % Positive 0% 6.3% 0% 43.8% 50% 100% 100% 50%It may be observed from Table 4 that analysis of samples collected fromsentinel animal yield relatively poor indication of parasites, detectingnone of the parasites by traditional screening and about 6.3% by PCR.Analysis of samples collected from the cage-level EAD collection mediaexhibit mixed success, identifying none of the parasites when thesentinel is not present within the cage but approximately 43.8% ofparasites when the sentinel is present. Analysis of samples collected byEAD collection media in-line with the exhaust airflow within the exhaustplenum exhibit excellent accuracy, detecting 100% of the parasitespresent. Analysis of samples collected by the EAD collection mediain-line with the exhaust airflow adjacent the pre-HEPA filter detected50% of the parasites present.

Estimated copy of the above parasites determined by real-time TaqMan PCRof collected EAD samples is illustrated in FIG. 8D. As noted above, theCt value is a relative measure of the concentration of a target. Aselevated concentration increases likelihood of detection, higher valuesof Ct are preferred. It may be observed from FIG. 8D that for eachparasite, the EAD samples collected by the EAD collection mediademonstrated Ct values comparable to or greater than swabbing.

These testing results indicate that PCR analysis of EAD samplescollected by EAD collection media in-line with exhaust airflow accordingto embodiments of the instant disclosure detect pathogens at a levelcomparable to or greater than traditional sentinels or EAD swabs. Theconcentration of sampled pathogens is also comparable to or greater thantraditional sentinels or EAD swabs.

Significantly, EAD collected by EAD collection media at the cage levelwas a relatively poor indicator of pathogens when a sentinel animal isnot present in the cage. However, the accuracy of these samplesincreased significantly when a sentinel animal is present within thecage. It is believed that this difference is the result of dustagitation provided by the moving animal.

To explore the anticipated benefits of dust agitation, without requiringthe use of a sentinel animal, an EAD collection system 900, illustratedin FIG. 9, is further evaluated. The system 900 includes a reversiblysealable enclosure 902 including a chamber 904 adapted to receive ananimal cage 906, an air intake 910 coupleable with an air supply 912,and an air return 914 coupleable with a vacuum source 916. The system700 further includes an actuation device 720 and a plurality of EADcollection media 922 (e.g., 922 a, 922 b, 922 c).

The actuation device 920 is in mechanical communication with theenclosure 902 and operable to agitate the contents of a cage 906positioned within the chamber 904. The actuation device 920 may beconfigured to move the enclosure 904 horizontally, vertically, and/orrotationally, alone or in any combination, in order to release dustparticles from bedding 924 positioned within the cage 906. In furtherembodiments, the actuation device 920 may be an ultrasonic device.

The EAD collection media 922 are suitable for capturing EAD transportedby a flow of sterile air 926 directed through the enclosure 902. The EADcollection media 922 may EAD collection media 224, as discussed above.In an embodiment, one or more EAD collection media 922 a is positionedwithin the cage 906 (e.g., on a wall of the cage 906 or suspendedtherein). In another embodiment, one or more EAD collection media 922 bis positioned outside of the cage 906 and within the enclosure 902(e.g., on a wall of the enclosure 902 or suspended within the chamber904). In further embodiments, one or more EAD collection media 922 c ispositioned outside of the enclosure 902 (e.g., within an airflow pathwayin fluid communication with the vacuum source 916).

EAD collected using the system 900 may be further analyzed for detectionof pathogens according to the method 100, where the collection operation102 is performed as discussed above with respect to the system 900.Operations 104-114 of method 100 are otherwise unchanged.

All references throughout this application, for example patent documentsincluding issued or granted patents or equivalents; patent applicationpublications; and non-patent literature documents or other sourcematerial; are hereby incorporated by reference herein in theirentireties, as though individually incorporated by reference, to theextent each reference is at least partially not inconsistent with thedisclosure in this application (for example, a reference that ispartially inconsistent is incorporated by reference except for thepartially inconsistent portion of the reference).

The terms and expressions which have been employed herein are used asterms of description and not of limitation, and there is no intention inthe use of such terms and expressions of excluding any equivalents ofthe features shown and described or portions thereof, but it isrecognized that various modifications are possible within the scope ofthe invention claimed. Thus, it should be understood that although thepresent invention has been specifically disclosed by preferredembodiments, exemplary embodiments and optional features, modificationand variation of the concepts herein disclosed may be resorted to bythose skilled in the art, and that such modifications and variations areconsidered to be within the scope of this invention as defined by theappended claims. The specific embodiments provided herein are examplesof useful embodiments of the present invention and it will be apparentto one skilled in the art that the present invention may be carried outusing a large number of variations of the devices, device components,methods steps set forth in the present description. As will be obviousto one of skill in the art, methods and devices useful for the presentmethods can include a large number of optional composition andprocessing elements and steps.

All patents and publications mentioned in the specification areindicative of the levels of skill of those skilled in the art to whichthe invention pertains. References cited herein are incorporated byreference herein in their entirety to indicate the state of the art asof their publication or filing date and it is intended that thisinformation can be employed herein, if needed, to exclude specificembodiments that are in the prior art. For example, when composition ofmatter are claimed, it should be understood that compounds known andavailable in the art prior to Applicant's invention, including compoundsfor which an enabling disclosure is provided in the references citedherein, are not intended to be included in the composition of matterclaims herein.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural reference unless thecontext clearly dictates otherwise. Thus, for example, reference to “acell” includes a plurality of such cells and equivalents thereof knownto those skilled in the art, and so forth. As well, the terms “a” (or“an”), “one or more” and “at least one” can be used interchangeablyherein. It is also to be noted that the terms “comprising”, “including”,and “having” can be used interchangeably. The expression “of any ofclaims XX-YY” (wherein XX and YY refer to claim numbers) is intended toprovide a multiple dependent claim in the alternative form, and in someembodiments is interchangeable with the expression “as in any one ofclaims XX-YY.”

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. Nothing herein is to be construed as anadmission that the invention is not entitled to antedate such disclosureby virtue of prior invention.

Every formulation or combination of components described or exemplifiedherein can be used to practice the invention, unless otherwise stated.

Whenever a range is given in the specification, for example, atemperature range, a time range, or a composition or concentrationrange, all intermediate ranges and sub-ranges, as well as all individualvalues included in the ranges given are intended to be included in thedisclosure. As used herein, ranges specifically include the valuesprovided as endpoint values of the range. For example, a range of 1 to100 specifically includes the end point values of 1 and 100. It will beunderstood that any sub-ranges or individual values in a range orsub-range that are included in the description herein can be excludedfrom the claims herein.

As used herein, “comprising” is synonymous with “including,”“containing,” or “characterized by,” and is inclusive or open-ended anddoes not exclude additional, unrecited elements or method steps. As usedherein, “consisting of” excludes any element, step, or ingredient notspecified in the claim element. As used herein, “consisting essentiallyof” does not exclude materials or steps that do not materially affectthe basic and novel characteristics of the claim. In each instanceherein any of the terms “comprising”, “consisting essentially of” and“consisting of” may be replaced with either of the other two terms. Theinvention illustratively described herein suitably may be practiced inthe absence of any element or elements, limitation or limitations whichis not specifically disclosed herein.

One of ordinary skill in the art will appreciate that startingmaterials, biological materials, reagents, synthetic methods,purification methods, analytical methods, assay methods, and biologicalmethods other than those specifically exemplified can be employed in thepractice of the invention without resort to undue experimentation. Allart-known functional equivalents, of any such materials and methods areintended to be included in this invention. The terms and expressionswhich have been employed are used as terms of description and not oflimitation, and there is no intention that in the use of such terms andexpressions of excluding any equivalents of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the invention claimed.Thus, it should be understood that although the present invention hasbeen specifically disclosed by preferred embodiments and optionalfeatures, modification and variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention as defined by the appended claims.

1. A method for detecting pathogens, comprising: receiving a test samplecomprising environmental air dust captured from airflow within anexhaust plenum of an individually ventilated cage rack (IVR) by acollection media, wherein a surface of the collection media is orientedat an acute angle with respect to a direction of airflow within theexhaust plenum during capture of the dust sample; isolating a pluralityof nucleic acids from the test sample, wherein the plurality of nucleicacids is representative of one or more pathogens; amplifying at leastone of the plurality of nucleic acids; and analyzing the amplifiednucleic acids to identify the presence or absence of a pathogen.
 2. Themethod of claim 1, wherein amplifying at least one of the plurality ofnucleic acids comprises at least one of loop mediated isothermicamplification and polymerase chain reaction (PCR).
 3. The method ofclaim 2, wherein amplifying at least one of the plurality of nucleicacids comprises PCR selected from the group consisting of endpoint PCRand real-time PCR.
 4. The method of claim 3, wherein: isolating theplurality of nucleic acids comprises extracting an RNA sample from thetest sample and reverse transcribing the extracted RNA sample into acDNA sample; amplifying at least one of the plurality of nucleic acidscomprises amplifying the cDNA sample by real-time PCR; and analyzing theamplified nucleic acids comprises measuring a Ct value of the amplifiedcDNA sample.
 5. The method of claim 3, wherein: isolating the pluralityof nucleic acids comprises extracting a DNA sample from the test sample;amplifying at least one of the plurality of nucleic acids comprisesamplifying the DNA sample by real-time PCR; and analyzing the amplifiednucleic acids comprises measuring a Ct value of the amplified DNAsample.
 6. The method of claim 3, wherein amplifying at least one of theplurality of nucleic acids comprises PCR and wherein analyzing theamplified nucleic acids comprises time of flight analysis of PCRproducts.
 7. The method of claim 1, wherein the test sample comprisesenvironmental air dust captured from the airflow over a time period ofat least 2 weeks.
 8. The method of claim 1, wherein the IVR furthercomprises at least one cage housing a test animal.
 9. The method ofclaim 8, wherein the test sample further comprises at least one of fecalpellets obtained from the test animal, biological material obtained froman oral swap of the test animal, biological material obtain from a bodyswab of the test animal, and tissue from the test animal.
 10. The methodof claim 9, wherein the one or more pathogens is selected from the groupconsisting of: Staphylococcus spp., Pasteurella spp., Proteus spp.,Klebsiella spp., Giardia spp., Cryptosporidium spp., Entamoeba spp.,Spironucleus spp., Murine norovirus, Pseudomonas spp., andbeta-hemolytic Streptococcus spp.
 11. The method of claim 1, whereinisolating the plurality of nucleic acids comprises at least one ofmagnetic isolation, column-based nucleic acid isolation, organicextraction methods, and alkaline lysis.
 12. The method of claim 1,wherein the IVR does not comprise a cage housing a sentinel animal. 13.The method of claim 1, wherein the collection media comprises a filterselected from the group consisting of mechanical filters, chemicalfilters, electrostatic filters, and wet scrubbers.
 14. The method ofclaim 13, wherein the collection media possesses an efficiency selectedwithin the range from 5% to 40%.
 15. The method of claim 13, wherein thecollection media is a graded filter.
 16. The method of claim 1, whereinthe angle of the collection media during capture of the dust sample isselected within the range from 15° to 25° with respect to the directionof airflow within the exhaust plenum.
 17. A method for detectingpathogens, comprising: receiving a test sample comprising environmentalair dust captured from airflow passing through an enclosure by acollection media, the enclosure comprising a chamber containing ananimal cage in fluid communication with the airflow and theenvironmental air dust released by agitation of soiled bedding of a testanimal positioned within the animal cage; isolating a plurality ofnucleic acids from the test sample, wherein the plurality of nucleicacids is representative of one or more pathogens; amplifying at leastone of the plurality of nucleic acids; and analyzing the amplifiednucleic acids to identify the presence or absence of a pathogen.
 18. Themethod of claim 17, wherein amplifying at least one of the plurality ofnucleic acids comprises at least one of loop mediated isothermicamplification and polymerase chain reaction (PCR).
 19. The method ofclaim 18, wherein amplifying at least one of the plurality of nucleicacids comprises PCR selected from the group consisting of endpoint PCRand real-time PCR.
 20. The method of claim 19, wherein: isolating theplurality of nucleic acids comprises extracting an RNA sample from thetest sample and reverse transcribing the extracted RNA sample into acDNA sample; amplifying at least one of the plurality of nucleic acidscomprises amplifying the cDNA sample by real-time PCR; and analyzing theamplified nucleic acids comprises measuring a Ct value of the amplifiedcDNA sample.
 21. The method of claim 19, wherein: isolating theplurality of nucleic acids comprises extracting a DNA sample from thetest sample; amplifying at least one of the plurality of nucleic acidscomprises amplifying the DNA sample by real-time PCR; and analyzing theamplified nucleic acids comprises measuring a Ct value of the amplifiedDNA sample.
 22. The method of claim 19, wherein amplifying at least oneof the plurality of nucleic acids comprises PCR and wherein analyzingthe amplified nucleic acids comprises time of flight analysis of PCRproducts.
 23. The method of claim 17, wherein the test sample comprisesenvironmental air dust captured from the airflow over a time period ofat least 2 weeks.
 24. The method of claim 17, wherein the environmentalair dust is released by agitation of the soiled bedding by the testanimal.
 25. The method of claim 17, wherein the environmental air dustis released by an actuation device in mechanical communication with theenclosure.
 26. The method of claim 25, wherein the enclosure does notcontain an animal during capture of the environmental air dust.
 27. Themethod of claim 17, wherein the test sample further comprises at leastone of fecal pellets obtained from the test animal, biological materialobtained from an oral swap of the test animal, biological materialobtain from a body swab of the test animal, and tissue from the testanimal.
 28. The method of claim 27, wherein the one or more pathogens isselected from the group consisting of: Staphylococcus spp., Pasteurellaspp., Proteus spp., Klebsiella spp., Giardia spp., Cryptosporidium spp.,Entamoeba spp., Spironucleus spp., Murine norovirus, Pseudomonas spp.,and beta-hemolytic Streptococcus spp.
 29. The method of claim 17,wherein isolating the plurality of nucleic acids comprises at least oneof magnetic isolation, column-based nucleic acid isolation, organicextraction methods, and alkaline lysis.
 30. The method of claim 17,wherein the collection media comprises a filter selected from the groupconsisting of mechanical filters, chemical filters, electrostaticfilters, and wet scrubbers.
 31. The method of claim 30, wherein thecollection media possesses an efficiency selected within the rangebetween 5% to 40%.
 32. The method of claim 30, wherein the collectionmedia is a graded filter.
 33. A system for collecting environmental airdust, comprising: a reversibly sealable enclosure, including: a chamberadapted to receive a cage for housing an animal, the cage being in fluidcommunication with the chamber; an air intake coupleable with an airsupply; and an air return coupleable with a vacuum source; wherein aportion of a flow of supplied air directed through the chamber, from theair supply to the air exhaust, passes through at least a portion of thecage when received in the chamber and is sufficient to transport aportion of environmental air dust contained within the received cage tothe air exhaust; an actuation device in mechanical communication withthe enclosure, the actuation device operable to agitate the contents ofthe cage when received within the chamber; and a collection mediasuitable for capturing at least a portion of the environmental air dusttransported by a flow of air directed through the chamber.
 34. Thesystem of claim 33, wherein the collection media is positioned withrespect to the enclosure such that the collection media impinges atleast a portion of the flow of air after passage through the cage. 35.The system of claim 33, wherein the collection media is positionedwithin the enclosure and outside of the cage.
 36. The system of claim33, wherein the collection media is positioned with respect to theenclosure such that the collection device impinges at least a portion ofthe flow of air after passage through the air return.
 37. The system ofclaim 33, wherein at least a portion of the collection media ispositioned on a wall of the cage.
 38. The system of claim 33, wherein atleast a portion of the collection media is suspended within the cage.39. The system of claim 33, wherein the collection media comprises afilter selected from the group consisting of mechanical filters,chemical filters, electrostatic filters, and wet scrubbers.
 40. Thesystem of claim 33, wherein the actuation device is operable toreversibly move the enclosure at least one of translationally androtationally with respect to an initial position.
 41. The system ofclaim 33, wherein the actuation device is an ultrasonic device.